Stroke is a major cause of death and disability and brain blood vessels have been implicated. Stroke outcome is worsened by endothelial cell (EC) apoptosis, failure of angiogenesis and insufficient growth of collateral vessels. In brain EC, sphingosine-1-phosphate (S1P) induces Akt/endothelial nitric oxide synthase (eNOS) activation, suppresses apoptosis and induces proliferation. Neurons, and possibly glial cells, are a source of S1P in ischemia because a S1P synthesizing enzymes (sphingosine kinase 2, SPK2) is upregulated in neurons following oxygen glucose deprivation (OGD) and middle cerebral artery occlusion in mice. We hypothesize that following cerebral ischemia, S1P exerts antiapoptotic and pro-angiogenic effects on EC. Three specific aims will test the hypothesis that S1P activates EC S1P1 receptors and the Akt/eNOS cascade, protecting EC from apoptosis and inducing neovascularization. Aim 1 will confirm and characterize the protective effect of S1P on EC after OGD, as well as the induction of proliferation in these cells. We propose that S1P1 receptors, Akt and eNOS play a key role in these effects. Aim 2 will extend preliminary findings showing that neurons can be a relevant source of S1P during stroke and that neuron-derived S1P could mediate the survival and proliferation of brain EC observed in Aim 1. With the use of specific inhibitors, and RNA interference technology, we will determine the role of the SPK2 subtype, and whether neuronal S1P acts mediates the above effects via S1P1 receptors. Aim 3 will examine the in vivo significance of SPK2 up-regulation and the activation of the S1P1/Akt/eNOS system following ischemia in mice. We hypothesize that administration of FTY720 (a pro-drug converted to a S1P receptor agonist by SPK2, but not SPK1) will protect EC function, enhance EC proliferation and induce neovascularization following stroke via SPK2, S1P1 and Akt/eNOS. This project will study a novel system that can provide long-lasting improvement in EC function following stroke and serve as a target for stroke therapy.